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Engineering New Functionality into Cas9
01 May 2019

Protein Engineering Applications for the Sony SH800 Cell Sorter

"Although the Cas9 molecule has democratized genome editing, it has deficiencies," says David Savage, PhD, Associate Professor of Biochemistry, Biophysics, and Structural Biology at the University of California, Berkeley. "One is that it is 'always on.' It's always out there trying to cut the genome and that can lead to off-target effects that might have therapeutic implications. So you really want to ensure that Cas9 cuts in the right cell at the right time." Dr. Savage's lab is engineering new functionality into Cas9 to overcome this and other challenges.

Developing Fluorescent Biosensors

The effort began when they were designing biosensors to quantitate metabolic phenotypes using fluorescence. "Imagine you want to make a yeast produce a gasoline molecule," he explains. "How do you know if you're successful? There is no easy, single-cell assay to quantitate gasoline." Instead, inspired by Nobel laureate Roger Tsien and others, they began engineering green fluorescent protein (GFP) into biosensing molecules. They found that "you can actually make allosterically regulated GFPs that can sense their local chemical environment."

"The SH800 was a revolution for us because it allowed us to do these very complicated, time-intensive flow cytometry experiments in our lab."

The lab set about creating allosterically engineered biosensors for various molecules using directed evolution and protein engineering. "The process inherently involved a lot of single-cell and fluorescence assays," Dr. Savage recalls. "It required a lot of time on a flow cytometer because there were screens and counterscreens. It's really difficult to do these experiments at a core facility, because we needed to use the instrument 24 hours a day, three times a week. But in the old days, having your own cell sorter was prohibitively complicated and expensive."

Then Sony Biotechnology introduced the SH800 cell sorter. "The SH800 was a revolution for us," Dr. Savage says, "because it allowed us to do these very complicated, time-intensive flow cytometry experiments right in our lab. That really opened the door." The lab ultimately created a platform of technologies for engineering allosteric metabolite biosensors.1

A Self-Regulatory Cas9 Molecule

Around the same time, CRISPR-Cas9 genome editing hit the mainstream. The team realized that their approach, which had worked so well with the biosensor project, could be used to engineer new functionality into Cas9. "In the same way that GFP could be turned into a biosensor, so could Cas9—but the output would be not GFP fluorescence but genome editing activity." In other words, Dr. Savage explains, "an allosterically regulated Cas9 molecule can be used to control the activity of Cas9 in a very directed fashion, so that you can make sure that Cas9 is on in the right place at the right time." Dr. Savage and his colleagues released their methodological platform for Cas9 engineering in 20142 and published a proof of concept in 2016.3

"In the same way that GFP could be turned into a biosensor, so could Cas9—but the output would be not GFP fluorescence but genome editing activity."

Recently, the lab has extended its Cas9 work to engineer sensitivity not just to small molecules but to other signals in the cell, including proteases—protein-cutting proteins commonly used in cell signaling and often by pathogens when invading cells.4 "A protease-sensitive Cas9, which we call ProCas9, can actually be a sentinel molecule to sense signaling pathways and viral infections inside a cell, and then respond to the situation, almost like an immune system," explains Dr. Savage. "And because it's Cas9, that response is completely programmable."

The programmability of ProCas9 has important biomedical and agricultural applications. "It opens up the ability to use Cas9 like an immune system, where it can sense an infection and then respond. That is actually how plant immune systems work. We showed that it works against common plant potyviruses. From a research perspective, you could do that same viral sensing and response in human cells too, because obviously viruses are important human pathogens. It combines the broad programmability of Cas9 with our discovery that protease recognition sites are also programmable."

Large Libraries, Progressive Sorting

The lab's Cas9 research relies heavily on their Sony SH800 sorter. The panels themselves are very simple, Dr. Savage explains. "Obviously, [the sorter] can process a wide range of different markers, but we tend to stick with genetically encoded fluorescent proteins—green and red, traditionally, and occasionally blue. Often we use an experimental color that we can track for quantitative changes, and a second or third color might be a control signal so that, when we see an experimental result, we know it's real. Truthfully, we don't do complicated analyses. What we do differently is to sort for very long periods of time. We could do an all-day, 12-hour sort to look at 10 million or more cells in very big libraries."

The second difference is that the lab often does multiple, progressive rounds of sorting—screens and counterscreens, as shown in Figure 1. "When we engineer allosteric proteins, they will change their function, in this case their fluorescence output, as a function of metabolic state. So as we either add the molecule to the culture or remove it, we can look at whether they are on when they're supposed to be on and off when they're supposed to be off. If you go back and forth, you can very quickly winnow down and find that needle in the haystack, the functional protein you were looking for all along. With the Sony SH800, we can go through multiple rounds of sorting. Without having to do much culturing, we can identify the protein variants we're looking for in an afternoon as opposed to over many weeks."

Transforming the Way Science Is Done

The Sony SH800 used by Dr. Savage, one of the first to be put into operation on the west coast, was originally selected for its ability to sort E. coli. "The range of fluidics chip sizes gives it very good fidelity for sorting individual bacterial cells," he says. "And their disposability makes the instrument more robust. We don't have to worry about cross-contamination or other fluidics issues. A traditional instrument might require additional cleaning and other maintenance. But we can just replace the chip, and it effectively becomes a new instrument."

Another bonus is its ease of use. "We do have a sort of super user," Dr. Savage says, "but it does not require an expert technician you would traditionally see at a core facility. Just as Cas9 is said to democratize genome editing, the SH800 democratizes cell sorting. Users still have to be well informed and diligent, but it really lowers the bar. Even graduate students or postdocs with just a few weeks of training are able to independently run experiments."

"Just as Cas9 is said to democratize genome editing, the Sony SH800 democratizes cell sorting."

For Dr. Savage and his colleagues, having a lab-based Sony SH800 has transformed their approach to science. "It changed the way we do experiments! It allowed us to do things we'd dreamed about but couldn't afford or physically manage. With the SH800, we could expand our libraries, do very long experiments, and run for days at a time in a way that we could never do at a core facility."

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